A Rapid Method for Detecting Normal or Modified Plant and Algal Carbonic Anhydrase Activity Using Saccharomyces cerevisiae

In recent years, researchers have attempted to improve photosynthesis by introducing components from cyanobacterial and algal CO2-concentrating mechanisms (CCMs) into terrestrial C3 plants. For these attempts to succeed, we need to understand the CCM components in more detail, especially carbonic anhydrase (CA) and bicarbonate (HCO3−) transporters. Heterologous complementation systems capable of detecting carbonic anhydrase activity (i.e., catalysis of the pH-dependent interconversion between CO2 and HCO3−) or active HCO3− transport can be of great value in the process of introducing CCM components into terrestrial C3 plants. In this study, we generated a Saccharomyces cerevisiae CA knock-out (ΔNCE103 or ΔCA) that has a high-CO2-dependent phenotype (5% (v/v) CO2 in air). CAs produce HCO3− for anaplerotic pathways in S. cerevisiae; therefore, the unavailability of HCO3− for neutral lipid biosynthesis is a limitation for the growth of ΔCA in ambient levels of CO2 (0.04% (v/v) CO2 in air). ΔCA can be complemented for growth at ambient levels of CO2 by expressing a CA from human red blood cells. ΔCA was also successfully complemented for growth at ambient levels of CO2 through the expression of CAs from Chlamydomonas reinhardtii and Arabidopsis thaliana. The ΔCA strain is also useful for investigating the activity of modified CAs, allowing for quick screening of modified CAs before putting them into the plants. CA activity in the complemented ΔCA strains can be probed using the Wilbur–Anderson assay and by isotope exchange membrane-inlet mass spectrometry (MIMS). Other potential uses for this new ΔCA-based screening system are also discussed.


Introduction
Carbonic anhydrases (CAs) catalyze the interconversion between CO 2 and bicarbonate (HCO 3 − ) in solutions [1]. Although the interconversion of CO 2 and HCO 3 − happens without a CA, it occurs at a very slow rate. CAs are essential for organisms to ensure they have a quick supply of CO 2 and HCO 3 − for various metabolic pathways. CAs also play a crucial role in photosynthesis. For example, the CO 2 -concentrating mechanisms (CCMs) of Chlamydomonas reinhardtii and cyanobacteria are powered by CAs [2][3][4][5]. In the biophysical CCMs of cyanobacteria and green algae, ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) is packaged in very specific compartments-carboxysomes for cyanobacteria and pyrenoids for green algae. The CCMs work to accumulate HCO 3 − to high levels in the cytosol (cyanobacteria) or chloroplast stroma (eukaryotic algae); then a specific CA is needed to convert the HCO 3 − to CO 2 for photosynthesis. This creates a local environment around Rubisco that has an elevated CO 2 concentration. In C. reinhardtii, the conversion of HCO 3 − to CO 2 is catalyzed by CAH3 in the thylakoid lumen inside the pyrenoid. Loss of this thylakoid CAH3 in C. reinhardtii results in very slow growth rates at ambient levels of CO 2 (~0.04% (v/v) CO 2 in air) [6]. Similarly, carboxysomal CAs in cyanobacteria are required for the conversion of accumulated HCO 3 − to CO 2 for fixation by Rubisco [7]. For photosynthetic organisms to function efficiently, the CAs must be in the correct interand intracellular locations. For example, cyanobacterial CAs inside the carboxysomes are critical for maintaining the CCM, but CA activity in the cytoplasm disrupts the CCM. Price et al. [8] showed that CA expression in the cytoplasm of Synechocystis cells caused the CCM to short-circuit.
In C 4 plants, the CCM is maintained by CA activity in mesophyll cells [9]. For C 4 plants, the first step of photosynthesis is the conversion of CO 2 that diffuses into the leaf mesophyll cells to HCO 3 − , which is catalyzed by a cytosolic CA [9]. DiMario et al. [10] demonstrated that the elimination of mesophyll cytoplasmic CA activity causes a reduction in photosynthesis for C 4 plants grown in ambient levels of CO 2 . C 3 plants, in contrast, do not have a CCM. However, C 3 plants still have a large number of genes encoding CA. In Arabidopsis, the α, β, γ, and γ-like isoforms of CA are encoded by 17 distinct genes [2]. The role of CAs in terrestrial C 3 plants is not well understood due to the compensatory effect of multiple isoforms. For example, DiMario, et al. [11] investigated the effects of knocking out the Arabidopsis CAs βCA2 and βCA4, which are present in the cytosol of leaf mesophyll cells, and observed that eliminating only one of the CAs resulted in no observable phenotype. When both βCA2 and βCA4 were knocked out, plants were unable to grow normally in low-CO 2 conditions. In addition, Medina-Puche et al. [12] and Hines et al. [13] observed that single knock-out lines for most βCAs in Arabidopsis had normal growth on air.
There have been attempts in recent years to improve photosynthesis by introducing CCM components from cyanobacteria, algae, or C 4 plants into terrestrial C 3 plants [ [14][15][16]. For these approaches to work, CAs must be modified and retargeted to specific locations in C 3 plants. This requires targeting CAs to the chloroplast thylakoid lumen or the cell wall. The CAs need to be modified and tagged to determine whether they are being targeted to the correct intracellular location. Thus, a rapid screen is needed to determine whether a protein modification inhibits CA activity. Transforming prospective CAs into plants is possible but requires significant time and resources. Heterologous complementation systems capable of detecting CA activity or active bicarbonate transport are valuable when studying CCM components in order to transform them into plants.
Here, we determined whether the high-CO 2 -dependent Saccharomyces cerevisiae CA knock-out line, ∆NCE103 (referred to here as ∆CA), is suitable as a heterologous complementation system for the detection of active CAs from plants and algae. The ∆CA strain cannot grow on ambient levels of CO 2 but can grow on high levels of CO 2 (5% CO 2 (v/v) in the air). The ∆CA strain lacks the gene NCE103, which encodes a single native S. cerevisiae CA [17]. It has been proposed that the loss of this native CA results in S. cerevisiae cells that do not have enough HCO 3 − for important metabolic processes such as fatty acid and nucleotide synthesis [18]. It has been speculated that S. cerevisiae requires some CA activity for survival at ambient levels of CO 2 because the uncatalyzed rate of CO 2 hydration to HCO 3 − produces insufficient HCO 3 − for anaplerotic pathways [19,20]. Aguilera et al. [18] hypothesized that ∆CA is not viable in ambient CO 2 largely because the cellular HCO 3 − level was insufficient for generating lipids. S. cerevisiae uses acetyl-CoA as a building block to synthesize neutral lipids (NL) such as triglycerides (TGs) and sterolesters (SEs) [21]. The first step in fatty acid biosynthesis is the carboxylation of acetyl-CoA to malonyl-CoA [22]. This reaction uses HCO 3 − generated from CO 2 by the native CA in the wild-type S. cerevisiae cell. Therefore, ∆CA can be used to detect and analyze the activity of normal or modified CAs and unusual CA-like proteins. To test this ∆CA-based complementation system, we first used ∆CA to investigate the activity of the human CA II (hCA) protein. We then tested the viability of tagged and codon-optimized hCA proteins to see if CA activity is affected by such modifications. We also tested the activity of CAs located in the mitochondria and thylakoids of C. reinhardtii. The mitochondrial CAs, CAH4 and CAH5, are β-CAs that are highly expressed in C. reinhardtii cells grown in ambient levels of CO 2 [23,24]. They have been shown to be necessary for optimal photosynthesis in cells grown in limiting-CO 2 conditions [25]. CAH3 is an α-CA located in the thylakoid lumen of C. reinhardtii that generates CO 2 for fixation by Rubisco inside the pyrenoid [26,27]. We also used an Arabidopsis thaliana β-CA called βCA3 to see if the ∆CA-based heterologous complementation system works for plant CAs. Aside from viability tests, CA activity was verified using the Wilbur-Anderson assay [28] and isotope exchange membrane-inlet mass spectrometry (MIMS). The results presented in this study suggest that the ∆CA strain can be used to determine the activity of CAs from different sources, as well as CAs that have been modified with tags and codon optimization.

The S. cerevisiae Strain ∆CA Has a High-CO 2 -Dependent Growth Phenotype
The CO 2 level requirement of ∆CA was characterized by conducting growth assays on solid media supplemented with different levels of CO 2 : 5% (v/v) CO 2 in air, 1% (v/v) CO 2 in air, and ambient CO 2 (~0.04% (v/v) CO 2 in the air) ( Figure 1). For these experiments, ∆CA-EV refers to the ∆CA strain transformed with an empty vector (EV) containing a selectable gene. For the positive control, ∆CA was transformed with a vector containing the S. cerevisiae NCE103 gene so that it expresses the native CA (∆CA-ScCA). Unlike the positive control, ∆CA only grows at 5% CO 2 and dies at 1% and ambient CO 2 . These results agree with an early report by Aguilera et al. [18]. To investigate the biochemical deficiency underlying the high-CO 2 -dependent phenotype of ∆CA, we incorporated radiolabeled 14 C-acetic acid into ∆CA-EV, ∆CA-ScCA, and ∆CA-hCA-YCO (∆CA complemented with a S. cerevisiae codon-optimized (YCO) version of hCA) for one hour in ambient-CO 2 conditions and assayed 14 C incorporation into lipids using a silicone oil filtering centrifugation assay. The incorporation of radiolabeled 14 C in neutral lipids was higher in cells reconstituted with hCA compared to ∆CA-EV after one hour. Furthermore, ∆CA-hCA-YCO had twice the incorporation of 14 C in neutral lipids compared to ∆CA after one hour ( Figure 2).

∆CA Can Be Used as a Heterologous Complementation System to Detect CA Activity of Normal and Modified CAs
The CO 2 growth requirement of ∆CA was used to characterize the CA activity of normal and modified CAs. hCA complemented the ∆CA phenotype in ambient CO 2 and 1% CO 2 (Figures 3 and 4) [29]. In the liquid growth assay, we observed that ∆CA-hCA-YCO grew faster than ∆CA-EV in ambient CO 2 . However, in both conditions, ∆CA-ScCA growth was faster compared to ∆CA-EV (Figure 3a,b). In the growth assay on solid media, ∆CA-hCA-YCO grew at a rate similar to ∆CA-ScCA in ambient CO 2 and 1% CO 2 ( Figure 4). Next, we transformed ∆CA with YCO hCA and Arabidopsis codon-optimized (Atex) hCA and checked their effect on CA activity. Additionally, we added the tags AcV5 and eGFP to ∆CA-hCA-YCO and ∆CA-hCA-Atex to see if they affected the growth of the S. cerevisiae. The growth assays on solid media show that the modified hCA variants complemented ∆CA in ambient CO 2 and 1% CO 2 ( Figure 5). We also compared the expression of hCA in the complemented lines by analyzing the protein's abundance via Western blots. In the strains complemented with the YCO genes (∆CA-hCA-YCO, ∆CA-hCA-YCO AcV5, and ∆CA-hCA-YCO eGFP), hCA expression was higher compared to strains complemented with Atex genes (∆CA-hCA-Atex, ∆CA-hCA-Atex AcV5, and ∆CA-hCA-Atex eGFP; Figure 6b). The protein expression of hCA was not affected by the addition of the AcV5 and eGFP     zero hours is shown as a negative control. 14 C incorporation was measured using a silicone oil filtering centrifugation assay. Bars represent means, and error bars represent standard errors (n = 3). Statistical significance among different groups was computed with ANOVA and Tukey's post hoc HSD test (p < 0.05), and different statistical groups are represented by the letters a and b above bars.

Plant and Algal Carbonic Anhydrases Show CA Activity in ΔCA-Based Heterologous Complementation System
To test the hypothesis that the ΔCA-based heterologous complementation system can rapidly detect the activity of different algal CA isoforms, we expressed the C. reinhardtii β-carbonic anhydrase CAH5 and α-carbonic anhydrase CAH3 in ΔCA. Expression of

Plant and Algal Carbonic Anhydrases Show CA Activity in ∆CA-Based Heterologous Complementation System
To test the hypothesis that the ∆CA-based heterologous complementation system can rapidly detect the activity of different algal CA isoforms, we expressed the C. reinhardtii β-carbonic anhydrase CAH5 and α-carbonic anhydrase CAH3 in ∆CA. Expression of CAH5 restored a normal growth phenotype in the ∆CA mutant when cells were grown in ambient CO 2 and 1% CO 2 (Figure 7). CAH5 protein expression was detected in ∆CA-CrCAH5 and in the positive control D66 (a wild-type C. reinhardtii strain) (Figure 8a). The full-length coding sequence (CDS) was used for the expression of CAH5 in ∆CA. The Western blot shows that S. cerevisiae was able to process the N-terminal mitochondrial sequence of CAH5 (Figure 8a). We observed a full-length polypeptide of 27.8 kD and a cleaved polypeptide of 20.4 kD, which is similar to the size observed in the positive control. This is the first report showing that CAH5 is an active CA in a heterologous system. The expression of CAH3 in ∆CA-CrCAH3-YCO restored growth in 1% CO 2 , but no growth was observed in ambient CO 2 (Figure 7). In the ∆CA-CrCAH3 strain containing the native C. reinhardtii CAH3 gene, growth was not restored in either limiting-CO 2 condition. The Western blot shows CAH3 expression in ∆CA-CrCAH3-YCO and in the positive control (Figure 8b). CrCAH5 and in the positive control D66 (a wild-type C. reinhardtii strain) (Figure 8a). The full-length coding sequence (CDS) was used for the expression of CAH5 in ΔCA. The Western blot shows that S. cerevisiae was able to process the N-terminal mitochondrial sequence of CAH5 (Figure 8a). We observed a full-length polypeptide of 27.8 kD and a cleaved polypeptide of 20.4 kD, which is similar to the size observed in the positive control. This is the first report showing that CAH5 is an active CA in a heterologous system. The expression of CAH3 in ΔCA-CrCAH3-YCO restored growth in 1% CO2, but no growth was observed in ambient CO2 (Figure 7). In the ΔCA-CrCAH3 strain containing the native C. reinhardtii CAH3 gene, growth was not restored in either limiting-CO2 condition. The Western blot shows CAH3 expression in ΔCA-CrCAH3-YCO and in the positive control (Figure 8b).
To test the activity of plant carbonic anhydrases in ΔCA, we transformed the S. cerevisiae mutant with the cytosolic carbonic anhydrase βCA3 from Arabidopsis. Normal growth was observed in ΔCA-AtβCA3 at all three CO2 levels ( Figure 9). This result suggests that βCA3 is an active CA.  plates and incubated at 30 • C in 5% CO 2 , 1% CO 2 , and ambient CO 2 (0.04%) for 3 days. The cells were standardized to an initial OD 600 of 0.1 and serially diluted before plating.  To test the activity of plant carbonic anhydrases in ∆CA, we transformed the S. cerevisiae mutant with the cytosolic carbonic anhydrase βCA3 from Arabidopsis. Normal growth was observed in ∆CA-AtβCA3 at all three CO 2 levels ( Figure 9). This result suggests that βCA3 is an active CA.  . ΔCA complemented with AtβCA3 can grow in limiting-CO2 conditions. ΔCA-ScCA, ΔCA-EV, and ΔCA-AtβCA3 cells were grown to logarithmic phase then plated in 10 µL spots on YM (his,-trp) plates and incubated at 30 °C in 5% CO2, 1% CO2, and ambient CO2 (0.04%) for 3 days. The cells were standardized to an initial OD600 of 0.01 and serially diluted before plating.

ΔCA-Based Heterologous Complementation System Can Be Used to Quantify CA Activity Using Wilbur-Anderson Assay and MIMS
To rapidly quantify the activity of normal or modified CAs in the ΔCA system, we used the Wilbur-Anderson assay and MIMS. The Wilbur-Anderson assay was successful in demonstrating the CA activity of ΔCA strains complemented with different variants of Figure 9. ∆CA complemented with AtβCA3 can grow in limiting-CO 2 conditions. ∆CA-ScCA, ∆CA-EV, and ∆CA-AtβCA3 cells were grown to logarithmic phase then plated in 10 µL spots on YM (-his,-trp) plates and incubated at 30 • C in 5% CO 2 , 1% CO 2 , and ambient CO 2 (0.04%) for 3 days. The cells were standardized to an initial OD 600 of 0.01 and serially diluted before plating.

∆CA-Based Heterologous Complementation System Can Be Used to Quantify CA Activity Using Wilbur-Anderson Assay and MIMS
To rapidly quantify the activity of normal or modified CAs in the ∆CA system, we used the Wilbur-Anderson assay and MIMS. The Wilbur-Anderson assay was successful in demonstrating the CA activity of ∆CA strains complemented with different variants of hCA (Table 1). CA activity was recorded as 4.7 ± 0.5 WAU mg −1 in ∆CA-hCA-YCO, which was the highest of all tested strains. The addition of AcV5 and eGFP decreased the CA activity to 3.9 ± 0.4 WAU mg −1 and 2.5 ± 0.3 WAU mg −1 , respectively. In the strains using Atex genes, CA activity was further reduced. The strain ∆CA-hCA-Atex showed CA activity around 1.9 ± 0.2 WAU mg −1 . Similar to the tagged YCO strains, the addition of AcV5 and eGFP decreased CA activity to 0.9 ± 0.1 WAU mg −1 and 1.4 ± 0.2 WAU mg −1 , respectively. CA activity was also measured in the ∆CA strains using MIMS ( Figure 10). We found that the cell lysate in ∆CA-hCA-YCO exhibited maximum CA activity. The AcV5 and eGFP tags reduced the CA activity significantly, consistent with measurements obtained using the Wilbur-Anderson assay. Table 1. The Wilbur-Anderson assay can be used to measure CA activity in the ∆CA mutant. The Wilbur-Anderson CA assay was performed on protein extracts from ∆CA strains grown in 5% CO 2 for 72 h. One WAU = (t 0 − t)/t where t 0 is the time for the uncatalyzed reaction and t is the time for the enzyme-catalyzed reaction. The negative control (∆CA-EV) has an activity of 0.9 ± 1 WAU mg −1 .  (Table 1). CA activity was recorded as 4.7 ± 0.5 WAU mg −1 in ΔCA-hCA-YCO, which was the highest of all tested strains. The addition of AcV5 and eGFP decreased the CA activity to 3.9 ± 0.4 WAU mg −1 and 2.5 ± 0.3 WAU mg −1 , respectively. In the strains using Atex genes, CA activity was further reduced. The strain ΔCA-hCA-Atex showed CA activity around 1.9 ± 0.2 WAU mg −1 . Similar to the tagged YCO strains, the addition of AcV5 and eGFP decreased CA activity to 0.9 ± 0.1 WAU mg −1 and 1.4 ± 0.2 WAU mg −1 , respectively. CA activity was also measured in the ΔCA strains using MIMS ( Figure 10). We found that the cell lysate in ΔCA-hCA-YCO exhibited maximum CA activity. The AcV5 and eGFP tags reduced the CA activity significantly, consistent with measurements obtained using the Wilbur-Anderson assay. Table 1. The Wilbur-Anderson assay can be used to measure CA activity in the ΔCA mutant. The Wilbur-Anderson CA assay was performed on protein extracts from ΔCA strains grown in 5% CO2 for 72 h. One WAU = (t0 − t)/t where t0 is the time for the uncatalyzed reaction and t is the time for the enzyme-catalyzed reaction. The negative control (ΔCA-EV) has an activity of 0.9 ± 1 WAU mg −1 .

Discussion
In this report, an S. cerevisiae CA knock-out strain (∆NCE103 or ∆CA) was utilized as a successful heterologous system for screening active carbonic anhydrases from plants and algae. Additionally, ∆CA has previously been reported as a potential tool for accelerating the discovery of non-sulfonamide-based CAIs (carbonic anhydrase inhibitors) for the treatment of CA-related diseases, such as glaucoma [30].
In this study, ∆CA was generated and found to have a high-CO 2 -dependent phenotype, meaning it requires high-CO 2 conditions to survive (Figure 1). This clear high CO 2 growth requirement indicates that ∆CA strains can be used for fast and accurate screening of CA activity or active bicarbonate transport. To confirm this hypothesis, we tested CAs from human red blood cells (hCA), Arabidopsis, and C. reinhardtii in the heterologous S. cerevisiae system. Along with rapidly screening for CA activity, ∆CA can also be used for the estimation of CA enzymatic activity using the Wilbur-Anderson assay and MIMS.
In mammals, CAs are expressed in almost all tissues and are involved in oxygen transport between lungs, red blood cells and tissues; pH regulation; ion exchange in the kidney; and electrical activity in the retina and nervous system [31][32][33]. Autotrophic organisms use CAs in CCMs, where CAs are involved in increasing inorganic carbon for carbon fixation [3]. In contrast, very little is known about the physiological role of CAs in heterotrophic microbes. According to Aguilera et al. [18], the CA-deficient S. cerevisiae mutant's need for elevated CO 2 concentrations originates from three bicarbonate-dependent carboxylation reactions catalyzed by pyruvate decarboxylase, acetyl-CoA carboxylase, and carbamoyl phosphate synthetase. These enzymes are involved in the synthesis of C 4 intermediates, fatty acids, arginine, and uracil, respectively [34]. These observations demonstrate that the S. cerevisiae CA is a key biosynthetic enzyme responsible for the viability of S. cerevisiae under aerobic conditions. Since ∆CA was complemented at air levels of CO 2 by the addition of hCA (Figures 3 and 4), we investigated whether fatty acid biosynthesis is bicarbonate-dependent at air levels of CO 2 (Figure 2). S. cerevisiae uses acetyl-CoA as a building block to synthesize neutral lipids (NL) such as triglycerides (TGs) and sterol-esters (SEs). Acetyl-CoA is first converted into malonyl-CoA by acetyl-CoA carboxylase, using HCO 3 − as a substrate. S. cerevisiae cells deficient in acetyl-CoA carboxylase are not able to make long chain saturated fatty acids for de novo growth [35]. To test our hypothesis, we introduced 14 C-acetic acid to ∆CA-ScCA, ∆CA-EV, and ∆CA-hCA grown on air levels of CO 2 for one hour. Acetic acid is rapidly converted into acetyl-CoA by acetyl-CoA synthetase (ACS2), which makes 14 C-acetic acid, a suitable radiolabeling substrate in S. cerevisiae. We observed that radiolabeled 14 C is incorporated into the chloroform-methanol fraction containing NLs. The incorporation of radiolabeled 14 C into NLs occurs at a higher count in ∆CA-hCA than in ∆CA-ScCA and ∆CA-EV ( Figure 2). This result confirmed that CAs produce HCO 3 − for the NL biosynthesis pathway. However, there are other bicarbonaterequiring pathways that are also involved in limiting the growth of ∆CA in air. Hence, if we introduce CAs or HCO 3 − transporters from plants and algae, they can help to increase the HCO 3 − pool required for different biological processes in the cell. This makes the ∆CA heterologous complementation system useful for identifying new bicarbonate transporters or CAs as suitable candidates to improve photosynthetic efficiency in C 3 crop plants.
This report shows that hCA displayed sufficient CA activity to rescue ∆CA grown on air levels of CO 2 (Figures 3 and 4). The results extend the work of Sangkaew et al. [30] who used this ∆CA system to screen CA inhibitors. To test modified CAs, S. cerevisiae codon-optimized (YCO) and Arabidopsis codon-optimized (Atex) hCA genes with added eGFP and AcV5 tags were used to complement ∆CA ( Figure 5. The hCA protein was detected in all the hCA variants, but the amount of protein was highest in S. cerevisiae codon-optimized hCA (Figure 6a,b). The different variants of hCA complement ∆CA even though the protein content differs in the strains. This relates to the concept that only a low amount of CA activity is needed to maintain vital biological functions in S. cerevisiae since CA is such a fast enzyme. This concept is also supported by plant studies showing that the majority of CA activity within the plant needs to be removed in order to observe a growth phenotype [10]. Although all the hCA variants rescued ∆CA, CA activity was highest in the strain using S. cerevisiae codon-optimized hCA (Table 1 and Figure 10). The low CA activity in Atex strains might also be because of low protein expression (Figure 6a,b). In terms of the effect of added tags, the MIMS and Wilbur-Anderson assay data showed that the addition of longer tags resulted in a larger reduction in CA activity. The hCA tagged with eGFP has lower CA activity compared to the hCA tagged with AcV5 (Table 1 and Figure 10). These results suggest that the ∆CA system can be used to rapidly test the suitability of carbonic anhydrases before introducing them into C 3 plants . Tags such as eGFP and AcV5 are widely used to determine the subcellular location of CAs, but there is a possibility that these tags can affect the functionality of the proteins. Thus, the ∆CA-based heterologous complementation system provides a rapid pipeline for the systematic assessment of normal and modified CAs before introducing them into C 3 plants. Mathematical models predict that installing a CCM into C 3 plants could improve leaf CO 2 uptake by up to 60% [36,37]. Hence, using ∆CA in conjugation with the Wilbur-Anderson assay and MIMS can shorten the process of selecting suitable CCM components from cyanobacteria and algae to transform into terrestrial C 3 plants.
To check the CA activity from an algal system, we expressed the β-carbonic anhydrase, CAH5, and the α-carbonic anhydrase, CAH3, from C. reinhardtii in ∆CA. CAH5 is present in the mitochondrial matrix and is required to maintain optimal rates of photoautotrophic growth on ambient levels of CO 2 [25]. CAH5 restores the growth of ∆CA at air levels of CO 2 and 1% CO 2 , but the rescued phenotype is weak compared to ∆CA-ScCA (Figure 7). This might indicate that CAH5 is a low-activity CA. Mitochondrial CA in C. reinhardtii is encoded by two genes (CAH4 and CAH5). This genetic redundancy could help the cell increase the amount of CA in the mitochondria since the enzyme has low activity, but it seems wasteful to produce large amounts of a protein with low activity rather than making a protein with high activity. The α carbonic anhydrase CAH3 was also tested in ∆CA. It is located in the thylakoid lumen in C. reinhardtii and has also been identified as an important component to maintain the CCM at low CO 2 levels [38,39]. It was previously reported that CrCAH3 is different from other α carbonic anhydrases given that it has an optimum CA activity at lower pH values than CAs of the same type, which normally operate at pH 7.0 and higher [6,40]. S. cerevisiae codon-optimized CAH3 restored the normal growth phenotype in ∆CA at 1% CO 2 but not at air levels, indicating that CAH3 activity might have been reduced because the S. cerevisiae cytoplasmic pH is around 7 (Figure 7). The Western blot clearly shows that CrCAH3 is produced in ∆CA (Figure 8b). The chloroplast transit peptide was removed from CrCAH3. The low activity could also be attributed to our use of a truncated version of CAH3 in ∆CA, although the protein length we used was reported to be the mature protein size [6,26]. Another possibility is that CAH3 requires post-translational modifications. Blanco-Rivero, et al. [41] reported that kinase activity is needed to activate CAH3 inside the lumen. The complementation of CrCAH5 and CrCAH3 (both YCO) in ∆CA suggests that the ∆CA-based heterologous complementation system can detect active CA enzymes from algal systems.
In general, α-carbonic anhydrases are structurally simpler than β-carbonic anhydrases and often have high specific activity. Humans only have α-carbonic anhydrases, while plants and algae have a wide variety of carbonic anhydrase families from α, β, γ, and θ classes. These CAs are localized to different intercellular and intracellular locations. Hence, the ∆CA heterologous complementation system could also be used as a tool to differentiate activity between CA families, which could help researchers select better CA candidates to integrate into C 3 crop plants.
To check if ∆CA is rescued by a plant's CA, we used β carbonic anhydrase βCA3, which localizes in the cytosol of Arabidopsis. βCA3 rescued ∆CA on 1% CO 2 and air levels of CO 2 , showing that it is an active CA enzyme (Figure 9).
After transforming crop plants with a protein, the main challenge occurs upon trying to determine if the protein is functional once it is correctly localized. The addition of a fluorescent tag reveals if the protein is in the correct location, but it cannot report if the protein is in the correct orientation in the membrane or if it is functional. In addition, when GFP is added to a protein, the protein is modified because an entire second protein has been attached. This modification could potentially alter the CAs' activity. The heterologous ∆CA-based system is an important tool for rapidly checking the activity of normal or modified CAs before integrating them into C 3 crop plants to improve photosynthetic efficiency (Figure 11a,b). The ∆CA system can also check the activity of proteins retargeted to different compartments in plants. Figure 11. Model proposing the use of ∆CA-based heterologous complementation systems to detect CA activity. In (a) the importance of carbonic anhydrase (CA) for the growth of S. cerevisiae at ambient CO 2 is shown and (b) discusses the pipeline to use ∆CA as a useful tool for rapidly detecting CA activity before introducing normal or modified CAs in crop plants.

S. cerevisiae Strain and Growth Conditions
Saccharomyces cerevisiae strain DDY2 (S. cerevisiae W303-1a diploid variant) was used as the starting stock for the generation of a CA knock-out (∆NCE103). Yeast minimal media (YM) supplemented with 6.7 g/L yeast nitrogen base, 20% (w/v) dextrose, and an amino acid mix was used to grow liquid cultures of the S. cerevisiae cells. YM plates were made by adding 1.5% (w/v) agar to liquid YM. The ∆CA generated in this study was grown in 5% (v/v) CO 2 in air at 30 • C, unless otherwise stated. Liquid cultures were grown on a rotary shaker at 30 • C in 5% (v/v) CO 2 in air and ambient CO 2 (0.04% (v/v) CO 2 in air). S. cerevisiae cells were grown on YM plates in three different CO 2 conditions: 5% (v/v)

S. cerevisiae CA Knock-Out Transformation
The plasmid MG0515 containing hCA-YCO, hCA-YCO AcV5, hCA-Atex, hCA-Atex AcV5, and CrCAH3-YCO, the plasmid MGO528 containing hCA-YCO eGFP, and hCA-Atex eGFP, and the plasmid pDD506 containing CrCAH5, CrCAH3, and AtβCA3 were transformed in E. coli TOP10 cells (One Shot™ TOP10 Chemically Competent E. coli). The plasmids were extracted from the transformed E. coli cultures using a GeneJET Plasmid Miniprep Kit (Thermo Scientific™) according to manufacturer's instructions. The plasmids were transformed in the ∆NCE103 mutant using a S. cerevisiae transformation protocol as described by Gietz and Schiestl [45]. The positive colonies were screened by colony PCR using primers complementary to the genes (Table S1).

High-CO 2 -Dependence Growth Assay
S. cerevisiae cell cultures were initiated from −80 • C glycerol stocks. The liquid cultures were grown to log phase in liquid YM in 5% CO 2 at 30 • C. The cultures were reinoculated for the growth assay and grown in 5% CO 2 and ambient CO 2 . The optical density at 600 nm (OD 600 ) of the cultures was adjusted to an initial OD 600 of 0.01. Relative growth rates were measured in liquid YM by monitoring the cultures' OD 600 using a spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). For the measurement of relative growth rates on solidified YM, the OD 600 of the cultures was standardized to 0.01, and 10 µL of serial dilutions were spotted onto YM plates. The plates were incubated at 30 • C in 5%, 1%, and ambient CO 2 for 72 h. The plates were photographed after 72 h.

Immunological Detection of Expressed Proteins in S. cerevisiae
Membrane-enriched protein fractions of S. cerevisiae were prepared according to Zhang et al. [46]. Proteins from the S. cerevisiae cell lysate were mixed with 2× Laemmli sample buffer and β-mercaptoethanol and heated to 95 • C for~7 min prior to resolution by SDS-PAGE on 12% (v/v) polyacrylamide gels (Mini-PROTEAN TGX, Bio-Rad Laboratories, Hercules, CA, USA). The expression of tagged proteins was detected immunochemically after transferring the proteins to a PVDF membrane. The membrane was blocked in TTBS (TBS with 0.1% (v/v) Tween) with 1% (w/v) bovine serum albumin at 4 • C overnight. The membrane was treated with monoclonal antibodies for 1 h at room temperature with the following dilutions: 1:1000 for hCA (Sigma-Aldrich, Darmstadt, Germany), 1:20,000 for CrCAH5 (Agrisera, Vännäs, Sweden) and 1:10,000 for CrCAH3 (Agrisera). The anti-CrCAH3 antibody is commercially available at Agrisera and the anti-CrCAH5 antibody was custom made by Agrisera. The membrane was treated with secondary anti-rabbit antibody (HRP conjugated from Bio-Rad) for 1 h at room temperature with the following dilutions: 1:500 for hCA (Sigma-Aldrich, Darmstadt, Germany) and 1:4000 for CrCAH5 and CrCAH3. Antibody binding was visualized by fluorescence detection with Thermo Scientific™ Pierce™ ECL Western Blotting Substrate on a Chemi-Doc XRS (Bio-Rad, Hercules, CA, USA).

Analysis of Neutral Lipid Synthesis
Using Radiolabel 14 C-Acetic Acid 14 C-acetic acid was incorporated in S. cerevisiae using the protocol described by Rogers and Henne [22], except that 50 µL of radiolabeling media was added to 1 mL of cell suspension (final 14 C-acetic acid concentration = 1.25 µCi/mL). Here, a 1 h radiolabeling pulse incubation was used to label neutral lipid species in ∆CA, ∆CA-ScCA and ∆CA-hCA-YCO.

Carbonic Anhydrase Activity Assay
CA activity was measured by the Wilbur-Anderson assay according to Mitra et al. [47]. The isotope exchange membrane-inlet mass spectrometry (MIMS) technique was used to measure CA activity as described by Price and Badger [48]. Briefly, 20 µL of S. cerevisiae cell lysate was added to a temperature controlled, 2 mL reaction cuvette connected to the inlet of a Finnegan DELTA-V Isotope Ratio Mass Spectrometer (Thermo Fisher Scientific). The 2 mL CA assay consisted of 100 mM HEPES-KOH (pH 7.4), 5 mM dithiothreitol, and 13 C 18 O 2 . The enhanced rate of 18 O exchange between 13 C 18 O 2 and H 2 16 O was calculated as a ratio between the increase in 18 O loss from 13 C 18 O 2 in the presence of CA compared to the uncatalyzed rate. Three technical replicates were run at 25 • C for each CA enzyme tested [10].
Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/plants11141882/s1; Figure S1: Plasmids pDD506(HIS3), MGO515(HIS3), and MGO528(URA3) were used in this study for the overexpression of proteins in S. cerevisiae, Figure S2: Relative intensity of the modified hCA bands for the immunoblot experiment shown in Figure 6a,b; Table S1: Primers used in this study for cloning.